1. Field of the Invention
The present invention relates to a method of producing ubiquinone-10 using a plant, a transgenic plant for use in the production method, and an expression cassette for use in the production of the transgenic plant.
2. Description of the Related Art
Ubiquinone is a component of the electron transfer system and is a compound which has a quinone backbone with an isoprenoid (a naturally-occurring organic compound whose basic unit is isopentenyl diphosphate (IPP) having 5 carbon atoms). Ubiquinone is a biological component which has a structure of 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone and plays an important role, which is called a coenzyme Q (CoQ). Naturally-occurring ubiquinones include homologs: ubiquinone-5 to ubiquinone-12, depending on the number of side-chain isoprenoid units.
Ubiquinone is present in animal and plant tissue, and microorganisms, in which it plays a physiological and biochemical important role. Ubiquinone is a vitamin-like substance which is also known as an antioxidant substance, and is conformed to have a pharmacological effect for skin aging, immunodeficiency, heart disease, periodontal disease, chronic fatigue syndrome, retrograde brain, neural motor function disorder, diabetes, stroke, arteriosclerosis, cancer, hypertension, hypotension, cerebral infarction, allergic disease and the like. Ubiquinone also has a pharmacological effect to enhance the ability of the heart, and specifically, has a beneficial effect for congestive cardiac failure, and in addition, palpitation/shortness of breath, foot or face swelling, oversensitivity to cold and the like (see Makoto Kawamukai, “Yubikinon no seigosei to atarashii seirikinou [Biosynthesis of ubiquinone and its new physiological function]”, Kagaku to Seibutsu [Chemistry and biology], 40 (5); 172-178; Makoto Kawamukai, J. Biosci. Bioeng. 94, 511-517 (2002); and Okada et al., Eur. J. Biochem. 255, 52-59 (1998) for reviews of ubiquinone).
Among the ubiquinones, only ubiquinone-10 (UQ-10) has been confirmed to have a pharmaceutical effect. Ubiquinone-10 is synthesized in the body, however, its amount is decreased due to aging, fatigue, stress or the like, and therefore, needs to be often supplied. Therefore, at present time, ubiquinone-10 is contained in not only medicaments but also cosmetics and sports drinks, and is also sold as an ingredient of health food and dietary supplement.
Ubiquinone-10 is currently extracted and purified from plants in the family Solanaceae, yeast or microorganisms. However, the production does not keep up with demand. There is a need for a production method in addition to the conventional techniques.
Isoprenoid which forms a side-chain of ubiquinone is a naturally-occurring organic compound whose basic unit is isopentenyl diphosphate (IPP) having 5 carbon atoms. A large amount of isoprenoid is present in nature. In addition to that, isoprenoid is used as a side-chain of ubiquinone, isoprenoid is known as a part of carotenoids, natural rubbers, or prenylated proteins. Isoprenoid is gradually synthesized into a long chain by binding a new IPP to a basic IPP or FPP (farnesyl diphosphate) during biosynthesis in organisms.
It is known that a decaprenyl diphosphate synthase gene (SEQ ID NO. 5), which is cloned from a bacterium Gluconobacter suboxydans, produces ubiquinone-10 when expressed in E. coli (Japanese Laid-Open Publication No. 10-57072; and Okada et al., Eur. J. Biochem. 255, 52-59 (1998)). However, when ubiquinone-10 is produced in a bacterium, such as E. coli, production of dietary supplements, foods, beverages, food additives and the like which contain ubiquinone-10, requires enormous cost and effort to purify ubiquinone-10. In contrast, when ubiquinone-10 is expressed in an edible plant, the cost and effort for the purification can be reduced. However, there has been no report that the above-described bacterial gene is expressed in plants to produce ubiquinone-10.
On the other hand, an attempt has been made to use a eukaryote (recombinant fission yeast) to produce ubiquinone-10 (Japanese Laid-Open Publication No. 9-173076). In Japanese Laid-Open Publication No. 9-173076, the decaprenyl synthase gene of the fission yeast S. pombe was cloned and sequenced, and a fusion gene linked with a mitchondrial targeting signal was used to transform the budding yeast S. cerevisiae. However, production of ubiquinone-10 in the recombinant yeast cell has not been confirmed. Moreover, it is suggested that enzyme activity is low in the recombinant yeast cell. In contrast, yeast expressing ddsA gene from Gluconobacter produced ubiquinone-10. Therefore, no technique of producing ubiquinone-10 using a recombinant cell except for E. coli and yeast has been established to date.
Therefore, there is a demand for a method of enhancing production of ubiquinone-10 using a transformed plant, and a plant having an enhanced amount of ubiquinone-10 production.
An object of the present invention is to provide a method of producing ubiquinone-10 in a transgenic plant, and a transgenic plant for producing ubiquinone-10. Another object of the present invention is to provide an expression cassette for use in preparation of the transgenic plant of the present invention. Still another object of the present invention is to provide a composition, a food additive, a supplement and a pharmaceutical composition which contain ubiquinone-10 produced by the plant.